Vibrio vulnificus is a pathogen that has been associated with septicemia and wound infection in patients with iron overload. Mortality rates often exceed 50%. Iron has been found to play a major role in the pathogenesis of V. vulnificus infections. We have cloned and sequenced the fur gene of V. vulnificus, a gene involved in the iron-regulatory control of virulence genes in V. cholerae and E. coli. coli. We hypothesize a virulence regulon exists in V. vulnificus that responds to iron. This proposal describes methods for identifying iron-regulated virulence determinants in V. vulnificus by using TnphoA mutagenesis to crete a series of insertion mutations in iron-regulated genes. The ability to use available host iron is an important determinant for pathogenicity in many bacteria. Virulence of V. vulnificus has been found to directly correlate with the ability to utilize transferrin as an iron source. We will use the transposon vector TnphoA to mutagenize cells and utilize streptonigrin enrichment to select for transferrin receptor mutants and analyze these mutants at the protein and genetic levels. A number of iron regulated virulence determinants in Vibrio species have complex regulation by iron. We will examine the mechanism of iron regulation in V. vulnificus in the TnphoA mutants. Additional regulatory factors involved in iron regulation will be isolated by examining for defects in normal regulation in a library of iron- regulated TnphoA mutants mutagenized with mini-transposons. The cloned transposon can be used as a probe to clone a cosmid that would complement the regulation defect. Elucidation of the mechanism of iron regulation in V. vulnificus should provide additional insight into the genetic control of bacterial virulence in this organism.